Temperature dependence of force, velocity, and processivity of single kinesin molecules

Using the bead assay in optical microscopy equipped with optical tweezers, we have examined the effect of temperature on the gliding velocity, force, and processivity of single kinesin molecules interacting with a microtubule between 15 and 35 degrees C. The gliding velocity increased with the Arrhenius activation energy of 50 kJ/mol, consistent with the temperature dependence of the microtubule-dependent ATPase activity. Also, the average run length, i.e., a measure of processivity of kinesin, increased on increasing temperature. On the other hand, the generated force was independent of temperature, 7.34 +/- 0.33 pN (average +/- S.D., n = 70). The gliding velocities decreased almost linearly with an increase in force irrespective of temperature, implying that the efficiency of mechano-chemical energy conversion is maintained constant in this temperature range. Thus, we suggest that the force generation is attributable to the temperature-insensitive nucleotide-binding state(s) and/or conformational change(s) of kinesin-microtubule complex, whereas the gliding velocity is determined by the ATPase rate.     

Effect of temperature on kinesin-driven microtubule gliding and kinesin ATPase activity

DeCuevas et al. [J. Cell Biol. 116 (1992) 957-965] demonstrated by circular dichroism spectroscopy for the kinesin stalk fragment that shifting temperature from 25 to 30 degrees C caused a conformational transition. To gain insight into functional consequences of such a transition, we studied the temperature dependence of a full-length kinesin by measuring both the velocity of microtubule gliding across kinesin-coated surfaces and microtubule-promoted kinesin ATPase activity in solution. The corresponding Arrhenius plots revealed distinct breaks at 27 degrees C, corroborating the temperature-dependent conformational transition for a motility-competent full-length kinesin. Microtubules were found to glide up to 45 degrees C; at higher temperatures, kinesin was irreversibly damaged.     

Speeding up kinesin-driven microtubule gliding in vitro by variation of cofactor composition and physicochemical parameters

So far, there has been a discrepancy between the velocities of kinesin-dependent microtubule motility measured in vitro and within cells. By changing ATP, Mg(2+), and kinesin concentrations, pH and ionic strength, we tried to find conditions that favour microtubule gliding across kinesin-covered glass surfaces. For porcine brain kinesin, we found that raising the molar Mg(2+)/ATP ratio can substantially elevate gliding velocity. Gliding became also faster after temperature elevation or lowering the number of kinesin molecules bound to the glass surface. The highest mean gliding velocity (1.8 microm/s+/-0.09 microm/s), approaching velocities measured for anterograde transport in vivo, was achieved by combination of favourable factors (2.5 m m ATP, 12.5 m m Mg(2+), 37 degrees C, 450 kinesin molecules/microm(2)).     

Biased binding of single molecules and continuous movement of multiple molecules of truncated single-headed kinesin

Conventional kinesin has a double-headed structure consisting of two motor domains and moves processively along a microtubule using the two heads cooperatively. The movement of single and multiple truncated heads of Drosophila kinesin was measured using a laser trap and nanometer detecting apparatus. Single molecules of single-headed kinesin bound to the microtubules with a 3.5 nm biased displacement toward the plus end of the microtubule. The position of these single-headed kinesin molecules bound to a microtubule did not change until they had dissociated, indicating that single kinesin heads utilize nonprocessive movement processes. Two molecules of single-headed kinesin moved continuously along a microtubule with a lower velocity and force than that of single molecules of double-headed kinesin. The biased binding of the heads determines the directionality of movement, whereas two molecules of single-headed kinesin move continuously without dissociation from a microtubule.     

Temperature dependence of the flexural rigidity of single microtubules

Although the flexural rigidity of a microtubule has previously been estimated by various methods, its temperature dependence has never been systematically examined. Here, we measured the flexural rigidity of a single taxol-stabilized microtubule from thermal fluctuation of the free end of a microtubule, the other end of which was fixed, at different temperatures; the results showed that the flexural rigidity is 2.54 × 10⁻²⁴ N m² independent of temperature in the range of 20-35 °C. Next, we applied temperature pulse microscopy (TPM) [K. Kawaguchi, S. Ishiwata, Thermal activation of single kinesin molecules with temperature pulse microscopy. Cell Motil. Cytoskeleton 49 (2001) 41-47; H. Kato, T. Nishizaka, T. Iga, K. Kinosita Jr., S. Ishiwata, Imaging of thermal activation of actomyosin motors. Proc. Natl. Acad. Sci. USA 96 (1999) 9602-9606], which created the temperature gradient (1-2 °C/μm) along a microtubule gliding on kinesins in the presence of ATP. As a result, the gliding microtubule was buckled between two interacting kinesin molecules, when the microtubule had been propelled faster by the rear kinesin (higher temperature) and slower by the front one (lower temperature). By estimating the critical force to induce buckling of a microtubule, the flexural rigidity of a microtubule was estimated to be (2.7-7.8) × 10⁻²⁴ N m², which was in good agreement with the value determined above. We discuss the buckling process based on the temperature dependence of the force-velocity relationship of kinesin motility.     

Pressure-Induced Changes in the Structure and Function of the Kinesin-Microtubule Complex

Kinesin-1 is an ATP-driven molecular motor that “walks” along a microtubule by working two heads in a “hand-over-hand” fashion. The stepping motion is well-coordinated by intermolecular interactions between the kinesin head and microtubule, and is sensitively changed by applied forces. We demonstrate that hydrostatic pressure works as an inhibitory action on kinesin motility. We developed a high-pressure microscope that enables the application of hydrostatic pressures of up to 200 MPa (2000 bar). Under high-pressure conditions, taxol-stabilized microtubules were shortened from both ends at the same speed. The sliding velocity of kinesin motors was reversibly changed by pressure, and reached half-maximal value at ∼100 MPa. The pressure-velocity relationship was very close to the force-velocity relationship of single kinesin molecules, suggesting a similar inhibitory mechanism on kinesin motility. Further analysis showed that the pressure mainly affects the stepping motion, but not the ATP binding reaction. The application of pressure is thought to enhance the structural fluctuation and/or association of water molecules with the exposed regions of the kinesin head and microtubule. These pressure-induced effects could prevent kinesin motors from completing the stepping motion.     

Fast vesicle transport in PC12 neurites: velocities and forces

Although the mechanical behavior of single-motor protein molecules such as kinesin has been carefully studied in buffer, the mechanical behavior of motor-driven vesicles in cells is much less understood. We have tracked single vesicles in neurites of PC12 cells with a spatial precision of ±30 nm and a time resolution of 120 ms. Because the neurites are thin, long, straight, and attached to the surface of planar cover glasses, the velocity of individual vesicles could be measured for times as long as 15 s and distances as long as 15 m. The velocity of anterograde vesicles was in most cases constant for periods of 1–2 s, then changed in a step-like fashion to a new constant velocity. The viscoelastic modulus felt by the vesicles within live PC12 cells was determined from the Brownian motion, using Masons generalization of the Stokes–Einstein equation. From Stokes law, the drag force at the smallest sustained velocity was 4.2±0.6 pN for vesicles of radius 0.30–0.40 m, about half the maximum force which conventional kinesin can develop during bead assays in buffer. We interpret the observed velocity steps as changes of ±1 or occasionally ±2 in the number of active motor proteins dragging that vesicle along a microtubule. Assuming that the motor is conventional kinesin, which hydrolyzes one ATP per 8 nm step along the microtubule, the motor protein efficiency in PC12 neurites is approximately 35%.     

ATPase kinetic characterization and single molecule behavior of mutant human kinesin motors defective in microtubule-based motility

Conventional kinesin is a microtubule-based motor protein that is an important model system for understanding mechanochemical transduction. To identify regions of the kinesin protein that participate in microtubule binding and force production, Woehlke et al. [(1997) Cell 90, 207-216] generated 35 alanine mutations in solvent-exposed residues. Here, we have performed presteady-state kinetic and single molecule motility analyses on three of these mutants [Y138A, loop 11 triple (L248A/D249A/E250A), and E311A] that exhibited a similar approximately 3-fold reduction in both microtubule gliding velocity and microtubule-stimulated ATPase activity. All mutants showed normal second-order ATP binding kinetics, indicating correct folding of the active site. The Y138A and loop 11 triple mutants were defective both in nucleotide hydrolysis and in microtubule-stimulated ADP release rates, the latter suggesting a defect in allosteric communication between the microtubule and the active site. A single molecule fluorescence assay further revealed that the loop 11 mutant is defective in initiating processive motion, suggesting that this loop is important for the initial contact between kinesin and the microtubule. Y138A, on the other hand, can bind to the microtubule normally but cannot move processively. For E311A, neither the rate of nucleotide hydrolysis nor ADP release could account for its slower ATPase and gliding velocity, which suggests that either phosphate release or a conformational transition is rate-limiting in this mutant. The single molecule assay showed that E311A has a reduced velocity of movement, but is not defective in processivity. Thus, while these mutants behave similarly in solution ATPase and multiple motor gliding assays, kinetic and single molecule analyses reveal defects in distinct processes in kinesin's mechanochemical cycle.     

Controlling kinesin by reversible disulfide cross-linking. Identifying the motility-producing conformational change

Conventional kinesin, a dimeric molecular motor, uses ATP-dependent conformational changes to move unidirectionally along a row of tubulin subunits on a microtubule. Two models have been advanced for the major structural change underlying kinesin motility: the first involves an unzippering/zippering of a small peptide (neck linker) from the motor catalytic core and the second proposes an unwinding/rewinding of the adjacent coiled-coil (neck coiled-coil). Here, we have tested these models using disulfide cross-linking of cysteines engineered into recombinant kinesin motors. When the neck linker motion was prevented by cross-linking, kinesin ceased unidirectional movement and only showed brief one-dimensional diffusion along microtubules. Motility fully recovered upon adding reducing agents to reverse the cross-link. When the neck linker motion was partially restrained, single kinesin motors showed biased diffusion towards the microtubule plus end but could not move effectively against a load imposed by an optical trap. Thus, partial movement of the neck linker suffices for directionality but not for normal processivity or force generation. In contrast, preventing neck coiled-coil unwinding by disulfide cross-linking had relatively little effect on motor activity, although the average run length of single kinesin molecules decreased by 30-50%. These studies indicate that conformational changes in the neck linker, not in the neck coiled-coil, drive processive movement by the kinesin motor.     

Mechanism of processive movement of monomeric and dimeric kinesin molecules

Kinesin molecules are motor proteins capable of moving along microtubule by hydrolyzing ATP. They generally have several forms of construct. This review focuses on two of the most studied forms: monomers such as KIF1A (kinesin-3 family) and dimers such as conventional kinesin (kinesin-1 family), both of which can move processively towards the microtubule plus end. There now exist numerous models that try to explain how the kinesin molecules convert the chemical energy of ATP hydrolysis into the mechanical energy to "power" their processive movement along microtubule. Here, we attempt to present a comprehensive review of these models. We further propose a new hybrid model for the dimeric kinesin by combining the existing models and provide a framework for future studies in this subject.     

Temperature dependence and Arrhenius activation energy of F-actin velocity generated in vitro by skeletal myosin.

The effect of temperature on the velocity of rhodamine phalloidin-labelled F-actin moving in vitro on rabbit skeletal myosin has been studied. Translating actin filaments were visualized by epi-fluorescence in an inverted microscope, equipped with temperature control (+/- 0.2 K) of the stage and objective. Images were recorded in real time at magnifications of 400x or 160x by an intensified CCD camera on video tape. Motion of individual filaments was tracked by hand and velocities determined using frame times recorded simultaneously on the video tape. Velocity changed from 12 microns per second at 42 degrees C to 11 nm per second at 3 degrees C. The Arrhenius plot is non-linear, with the data following a cubic regression curve with no evident breaks or jumps. Data taken over the temperature range from single preparations followed the same curve for both heating and cooling; this indicates reversibility and absence of hysteresis. A hyperbolic model that smoothly translates with temperature between two asymptotic activation energies fits the data above 7 degrees C: these energies are 50(+/- 5) kJ per mole (Q10 = 1.9) at high temperatures and 289(+/- 29) kJ per mole (Q10 = 76.5) at low temperature with a transition temperature of 15.4(+/- 0.6) degrees C. These values are compared with other measurements made in vitro, in solution studies and on muscle fibres. An Arrhenius activation energy of 50 kJ per mole and a transition temperature of 15 degrees C are consistent with previous determinations but 289 kJ per mole is significantly greater than has been seen at low temperatures in other systems. This may indicate a different rate-limiting step in the kinetics of skeletal myosin driving actin filaments in vitro below 15 degrees C. Current determinations of the myosin "step-size" assume that the actin velocity is determined by the rate of ATP hydrolysis; the data confirm similar activation energies above 20 degrees C but they show that the temperature dependencies and activation energies are different at lower temperatures, implying uncoupling of the two processes.     

Genetic evidence for a microtubule-destabilizing effect of conventional kinesin and analysis of its consequences for the control of nuclear distribution in Aspergillus nidulans

Conventional kinesin is a microtubule-dependent motor protein believed to be involved in a variety of intracellular transport processes. In filamentous fungi, conventional kinesin has been implicated in different processes, such as vesicle migration, polarized growth, nuclear distribution, mitochondrial movement and vacuole formation. To gain further insights into the functions of this kinesin motor, we identified and characterized the conventional kinesin gene, kinA, of the established model organism Aspergillus nidulans. Disruption of the gene leads to a reduced growth rate and a nuclear positioning defect, resulting in nuclear cluster formation. These clusters are mobile and display a dynamic behaviour. The mutant phenotypes are pronounced at 37 degrees C, but rescued at 25 degrees C. The hyphal growth rate at 25 degrees C was even higher than that of the wild type at the same temperature. In addition, kinesin-deficient strains were less sensitive to the microtubule destabilizing drug benomyl, and disruption of conventional kinesin suppressed the cold sensitivity of an alpha-tubulin mutation (tubA4). These results suggest that conventional kinesin of A. nidulans plays a role in cytoskeletal dynamics, by destabilizing microtubules. This new role of conventional kinesin in microtubule stability could explain the various phenotypes observed in different fungi.     

Effect of temperature on kinetics and differential mobility of empty and loaded nucleoside transporter of human erythrocytes

The transmembrane equilibration of [3H]uridine was measured in human erythrocytes as a function of temperature using rapid kinetic techniques. Arrhenius plots of the maximum velocity of equilibrium exchange were continuous between 5 and 30 degrees C (Ea = 17-20 kcal/mol), but the increase in velocity with increase in temperature leveled off above 30 degrees C. This leveling off did not reflect heat inactivation of the carrier since transport activity was stable for 3 h at 37 degrees C. Transmembrane equilibration of uridine in equilibrium exchange and zero-trans modes at 5, 15, 25, and 35 degrees C conformed to appropriate integrated rate equations derived for the simple transporter. The nucleoside transporter exhibited directional symmetry, but the loaded carrier moved on the average 5 times more rapidly than the empty carrier at 15, 25, and 35 degrees C, but 25-40 times faster at 5 degrees C. This marked shift in differential mobility of loaded and empty carrier between 15 and 5 degrees C was entirely attributable to an impairment of mobility of empty carrier. The Michaelis-Menten constant for equilibrium exchange increased about 3-fold with increase in temperature between 5 and 35 degrees C. The van't Hoff plot of the values was approximately linear and yielded an estimate of the enthalpy of carrier:substrate dissociation of 7.8 kcal/mol.     

Role of the kinesin neck linker and catalytic core in microtubule-based motility

Kinesin motor proteins execute a variety of intracellular microtubule-based transport functions [1]. Kinesin motor domains contain a catalytic core, which is conserved throughout the kinesin superfamily, followed by a neck region, which is conserved within subfamilies and has been implicated in controlling the direction of motion along a microtubule [2] [3]. Here, we have used mutational analysis to determine the functions of the catalytic core and the approximately 15 amino acid 'neck linker' (a sequence contained within the neck region) of human conventional kinesin. Replacement of the neck linker with a designed random coil resulted in a 200-500-fold decrease in microtubule velocity, although basal and microtubule-stimulated ATPase rates were within threefold of wild-type levels. The catalytic core of kinesin, without any additional kinesin sequence, displayed microtubule-stimulated ATPase activity, nucleotide-dependent microtubule binding, and very slow plus-end-directed motor activity. On the basis of these results, we propose that the catalytic core is sufficient for allosteric regulation of microtubule binding and ATPase activity and that the kinesin neck linker functions as a mechanical amplifier for motion. Given that the neck linker undergoes a nucleotide-dependent conformational change [4], this region might act in an analogous fashion to the myosin converter, which amplifies small conformational changes in the myosin catalytic core [5,6].     

短期高温胁迫对高产小麦品系灌浆后期旗叶光系统Ⅱ功能的影响

以叶绿素快相荧光动力学曲线(OJIP)为探针,探讨了高温胁迫对高产小麦品系01-35灌浆后期光系统Ⅱ（PSⅡ）功能的影响.结果表明,在37 ℃～43 ℃范围内,随温度升高Q_A还原程度和还原速率增大,至43 ℃时分别比室温下增加了23.89%和24.09%,表明Q_A→Q_B的电子传递受到抑制;43 ℃时PSⅡ的电子受体库降至室温下的47.4%,表明高温胁迫伤害了PSⅡ受体库;而PSⅡ供体侧未受到影响.当温度达到46 ℃时,Q_A还原程度和还原速率分别比室温下增加了13.95%和20.48%,但比43 ℃时显著下降,而PSⅡ电子受体库与43 ℃时相比无显著变化,表明46 ℃时PSⅡ供体侧受到伤害.与对照品种鲁麦14相比,高温胁迫下高产小麦的捕光色素复合体仍能捕获较多的光能,而且将捕获的光能更多的用于电子传递,表明高产小麦的捕光色素复合体及电子传递体耐受高温的能力较强,能够维持较高的电子传递能力.     

Single molecule processes on the stepwise movement of ATP-driven molecular motors

Movement is a fundamental characteristic of all living things. This biogenic function that is attributed to the molecular motors such as kinesin, dynein and myosin. Molecular motors generate forces by using chemical energy derived from the hydrolysis reaction of ATP molecules. Despite a large number of studies on this topic, the chemomechanical energy transduction mechanism is still unsolved. In this study, we have investigated the chemomechanical coupling of the ATPase cycle to the mechanical events of the molecular motor kinesin using single molecule detection (SMD) techniques. The SMD techniques allowed to detection of the movement of single kinesin molecules along a microtubule and showed that kinesin steps mainly in the forward direction, but occasionally in the backward. The stepping direction is determined by a certain load-dependent process, on which the stochastic behavior is well characterized by Feynman's thermal ratchet model. The driving force of the stepwise movement is essentially Brownian motion, but it is biased in the forward direction by using the free energy released from the hydrolysis of ATP.     

Kinesin velocity increases with the number of motors pulling against viscoelastic drag

Although the properties of single kinesin molecular motors are well understood, it is not clear whether multiple motors pulling a single vesicle in a cell cooperate or interfere with one another. To learn how small numbers of motors interact, microtubule gliding assays were carried out with full-length Drosophila kinesin in a novel motility medium containing xanthan, a stiff, water-soluble polysaccharide. At 2 mg/ml xanthan, the zero-shear viscosity of this medium is 1,000 times the viscosity of water, similar to cellular viscosity. To mimic the rheological drag force on the motors when attached to a vesicle in a cell, we attached a 2 μm bead to one end of the microtubule (MT). During gliding assays in our novel medium, the moving bead exerted a drag force of 4–15 pN on the kinesins pulling the MT. The velocity of MTs with an attached bead increased with MT length and with kinesin concentration. The increase with MT length arose because the number of motors is directly proportional to MT length. Our results show that small numbers of kinesins cooperate constructively when pulling against a viscoelastic drag. In the absence of a bead but still in the viscous medium, MT velocity was independent of MT length and kinesin concentration because the thin MT, like a snake moving through grass, was able to move between xanthan molecules with little resistance. A minimal shared-load model in which the number of motors is proportional to MT length fits the observed dependence of gliding velocity on MT length and kinesin concentration.     

Effect of temperature on the velocity of erythrocyte aggregation

The velocity of the aggregation of human erythrocytes was examined in the range of 5-43 degrees C with a rheoscope combined with a video camera, an image analyzer and a computer. (1) With increasing temperature, the velocity of erythrocyte aggregation induced by fibrinogen, immunoglobulin G and artificial macromolecules (dextran of 70 kDa and poly(glutamic acid) of 50 kDa) increased. However, the relationship between the velocity of erythrocyte aggregation and the temperature was different among these macromolecules. (2) In 70% autologous plasma, the velocity of erythrocyte aggregation was minimum at 15-18 degrees C, and increased at both higher and lower temperatures. (3) The shape of erythrocyte aggregates in 12 mumol/l fibrinogen (containing 770 mumol/l albumin) and in 70% autologous plasma was dependent on temperature: three-dimensional below 15-18 degrees C and one-dimensional (mainly rouleaux) above 15-18 degrees C. However, the shape of aggregates in 27 mumol/l immunoglobulin G (containing 770 mumol/l albumin) was three-dimensional in all temperature ranges. (4) The temperature dependency of erythrocyte aggregation was discussed in terms of the changes of medium viscosity, of erythrocyte properties and of bridging macromolecules.     

A three-domain structure of kinesin heavy chain revealed by DNA sequence and microtubule binding analyses

The structure and function of kinesin heavy chain from D. melanogaster have been studied using DNA sequence analysis and analysis of the properties of truncated kinesin heavy chain synthesized in vitro. Analysis of the sequence suggests the existence of a 50 kd globular amino-terminal domain that contains an ATP binding consensus sequence, followed by another 50-60 kd domain that has sequence characteristics consistent with the ability to fold into an alpha helical coiled coil. The properties of amino- and carboxy-terminally truncated kinesin heavy chains synthesized in vitro reveal that a 60 kd amino-terminal fragment has the nucleotide-dependent microtubule binding activities of the intact kinesin heavy chain, and hence is likely to be a "motor" domain. Finally, the sequence data indicate the presence of a small carboxy-terminal domain. Because it is located at the end of the molecule away from the putative "motor" domain, we propose that this domain is responsible for interactions with other proteins, vesicles, or organelles. These data suggest that kinesin has an organization very similar to that of myosin even though there are no obvious sequence similarities between the two molecules.     

Structure and dynamics of the Golgi complex at 15 degrees C: low temperature induces the formation of Golgi-derived tubules

Immunofluorescence and cryoimmunoelectron microscopy were used to examine the morphologic and functional effects on the Golgi complex when protein transport is blocked at the ERGIC (endoplasmic reticulum-Golgi intermediate compartment) in HeLa cells incubated at low temperature (15 degrees C). At this temperature, the Golgi complex showed long tubules containing resident glycosylation enzymes but not matrix proteins. These Golgi-derived tubules also lacked anterograde (VSV-G) or retrograde (Shiga toxin) cargo. The formation of tubules was dependent on both energy and intact microtubule and actin cytoskeletons. Conversely, brefeldin A or cycloheximide treatments did not modify the appearance. When examined at the electron microscope, Golgi stacks were long and curved and appeared connected to tubules immunoreactive to galactosyltransferase antibodies but devoid of Golgi matrix proteins. Strikingly, COPI proteins moved from membranes to the cytosol at 15 degrees C, which could explain the formation of tubules.